Treatment of arteriosclerosis and xanthoma

ABSTRACT

A combination of one or more HMG-CoA reductase inhibitors (for example pravastatin, lovastatin, simvastatin, fluvastatin, rivastatin or atorvastatin) with one or more insulin sensitizers (for example troglitazone, pioglitazone, englitazone, BRL-49653, 5-(4-{2- 1-(4-2&#39;-pyridylphenyl)ethylideneaminooxy!-ethoxy}benzyl)thiazolidine-2,4-dione, 5-{4-(5-methoxy-3-methylimidazo 5,4-b!pyridin-2-yl-methoxy)benzyl}thiazolidine-2,4-dione or its hydrochloride, 5- 4-(6-methoxy-l-methylbenzimidazol-2-ylmethoxy)benzyl!thiazolidine-2,4-dione, 5- 4-(l-methylbenzimidazol-2-ylmethoxy)benzyl!-thiazolidine-2,4-dione and 5- 4-(5-hydroxy-1,4,6,7-tetramethylbenzimidazol-2-ylmethoxy) benzyllthiazolidine-2,4-dione) exhibits a synergistic effect and is significantly better at preventing and/or treating arteriosclerosis and/or xanthoma than is either of the components of the combination alone.

BACKGROUND OF THE INVENTION

The present invention relates to methods and compositions for thetreatment and prophylaxis of arteriosclerosis and/or xanthoma.

Throughout the world, in recent years, the tendency has been for theincidence of coronary artery disease and arteriosclerosis, includingatherosclerosis, to increase, even in those countries in which hithertothey have not been prevalent. Amongst the factors implicated in such anincrease are changes in lifestyle, including the "Western" meat-richdiet, and the adoption of such a diet even in countries where it is nottraditional, and the general increase in the average age of thepopulation. As a result, these diseases and arteriosclerosis, inparticular, are widely feared as arteriosclerosis is a well knownpotential cause of unexpected death, for example by such sequelae ofarteriosclerosis as myocardial infarction.

One of the main risk factors implicated in these diseases is a highblood plasma lipid level, particularly a high blood plasma cholesterollevel. There have, therefore, been many attempts to use an agent whichlowers the cholesterol level in order to prevent and cure thesediseases, and many compounds have been developed which, to a greater orlesser extent, have this effect. For example, one such compound, whichhas been very successful and is very well known is pravastatin, which isa lipid regulating agent and is an inhibitor of3-hydroxy-3-methylglutaryl-coenzyme A reductase (hereinafter referred toas "HMG-CoA reductase inhibitor") which is believed to act on therate-determining step of cholesterol biosynthesis. It has been reportedthat coronary arteriosclerosis and xanthoma may be prevented in rabbitsreceiving pravastatin, but its efficacy remains insufficient Biochimicaet Biophysica Acta, 960, 294-302 (1988)!. Studies to control coronaryarteriosclerosis and xanthoma have been carried out using a combinationof two lipid regulating agents, pravastatin and cholestyramine, which iswell known as an agent for lowering lipoprotein levels, but the efficacyof this combination also remains insufficient Atherosclerosis, 83, 69-80(1990)!.

It has been proposed in Japanese Patent Kokai Application No. Hei7-41423 that a specific class of insulin resistance-improving agents,for example troglitazone, may be effective in the treatment andprophylaxis of arteriosclerosis, particularly atherosclerosis, but,again, the efficacy of such compounds is not quite satisfactory.

BRIEF SUMMARY OF INVENTION

We have now surprisingly found that the application of a combination ofone or more HMG-CoA reductase inhibitors with one or more insulinsensitizers exhibits a synergistic effect and is significantly better atpreventing and/or treating arteriosclerosis and/or xanthoma than iseither of the components of the combination alone. Indeed, employing thenew combination of the present invention, these diseases may be slowlybut steadily curable.

It is therefore an object of the present invention to provide acombination of one or more HMG-CoA reductase inhibitors with one or moreinsulin sensitizers or insulin resistance-improving agents.

It is a further, and more specific object of the invention to providesuch a combination exhibiting a synergistic effect.

It is a still further object of the invention to provide methods andcompositions using such a combination for the prevention and/ortreatment of arteriosclerosis and/or xanthoma.

Other objects and advantages of the present invention will becomeapparent as the description proceeds.

Thus, in a first aspect, the present invention consists in a method forthe prevention or treatment of arteriosclerosis or xanthoma, whichmethod comprises administering to a patient suffering from orsusceptible to arteriosclerosis or xanthoma a first agent selected fromthe group consisting of HMG-CoA reductase inhibitors and a second agentselected from the group consisting of insulin sensitizers, said firstand second agents being administered together or within such a period asto act synergistically together.

The invention also provides a packaged pharmaceutical formulation forthe treatment or prophylaxis of arteriosclerosis or xanthoma, comprisinga first agent selected from the group consisting of HMG-CoA reductaseinhibitors and a second agent selected from the group consisting ofinsulin sensitizers, said first and second agents being in admixture orpackaged separately.

In a still further aspect, the invention provides a pharmaceuticalcomposition for the treatment or prophylaxis of arteriosclerosis orxanthoma, comprising a first agent selected from the group consisting ofHMG-CoA reductase inhibitors and a second agent selected from the groupconsisting of insulin sensitizers.

DETAILED DESCRIPTION OF INVENTION

At present, the experimental evidence seems to us to suggest that thesynergistic effect arises from an interaction between the modes ofaction of the two classes of compounds, the HMG-CoA reductase inhibitorsand the insulin sensitizers, and so the chemical structure of thecompounds is believed to be of less importance than their activities.Accordingly, any compound having HMG-CoA reductase inhibitory activitymay be used as the first agent, whilst any compound having insulinsensitizing activity may be used as the second agent.

The HMG-CoA reductase inhibitors are commonly used for the treatment orprophylaxis of hyperlipemia, and may comprise naturally occurringsubstances which originate in the metabolism of microorganisms,semi-synthetic substances derived therefrom and totally syntheticsubstances. Of these compounds, examples of preferred compounds includepravastatin, lovastatin, simvastatin, fluvastatin, rivastatin andatorvastatin. Pravastatin is disclosed in Japanese Patent PublicationNo. Sho 61-13699 and in U.S. Pat. Nos. 4,346,227 and 4,448,979, and itsformula (as the sodium salt) is sodium1,2,6,7,8,8a-hexahydro-6,8-tetrahydroxy-2-methyl-1-naphthaleneheptanoate.Lovastatin is disclosed in Japanese Patent Kokai Application No. Sho58-16875 and in European Patent No. 22 478, and its formula is 6-{2-1,2,6,7,8,8a-hexahydro-8-(2-methylbutyryloxy)-2,6-dimethyl-1-naphthyl!ethyl}tetrahydro-4-hydroxy-2H-pyran-2-one.Simvastatin is disclosed in Japanese Patent Kokai Application No. Hei1-1476 and in European Patent No. 33 538, and its formula is6-{2-1,2,6,7,8,8a-hexahydro-8-(2,2-dimethylbutyloxy)-2,6-dimethyl-1-naphthyl!ethyl}tetrahydro-4-hydroxy-2H-pyran-2-one.Fluvastatin is disclosed in Japanese Patent Publication No. Hei 2-46031and in U.S. Pat. No. 4,739,073, and its formula (as the sodium salt) issodium 7-3-(4-fluoro-phenyl)-1-methylethyl)-1H-indol-2-yl)-3,5-dihydroxy-6-heptanoate.Rivastatin is disclosed in Japanese Patent Kokai Application No. Hei1-216974 and in U.S. Pat. Nos. 5,006,530, 5,169,857 and 5,401,746, andits formula (as the sodium salt) is sodium7-(4-fluorophenyl)-2,6-diisopropyl-5-methoxymethylpyridin-3-yl)-3,5-dihydroxy-6-heptanoate.Atorvastatin is disclosed in Japanese Patent Kokai Application No. Hei3-58967 and in U.S. Pat. No. 5,273,995, and its formula is2-(4-fluoro-phenyl)-5-(1-methylethyl)-3-phenyl-4-(phenylcarbamoyl)-1H-pyrrole-1-(2,4-dihydroxyhexanoic)acid.

The insulin sensitizer, the other active ingredient of the invention,may also be referred to as an insulin resistance-improving agent, andwas originally used for the prevention and/or treatment of diabetes. Theterm embraces a wide variety of compounds, typically thiazolidinedionecompounds, oxazolidinedione compounds and oxathiadiazole compounds.

These compounds are disclosed in, for example, Japanese Patent KokaiApplications No. Hei 4-69383 and Hei 7-330728, WO 89/08651, WO 91/07107,WO 92/02520, WO 94/01433, and U.S. Pat. Nos. 4,287,200, 4,340,605,4,438,141, 4,444,779, 4,461,902, 4,572,912, 4,687,777, 4,703,052,4,725,610, 4,873,255, 4,897,393, 4,897,405, 4,918,091, 4,948,900,5,002,953, 5,061,717, 5,120,754, 5,132,317, 5,194,443, 5,223,522,5,232,925, 5,260,445 and European Patent No. 676 398 etc. Of these,examples of preferred compounds include troglitazone, pioglitazone,englitazone, BRL-49653, 5-(4-{2-1-(4-2'-pyridylphenyl)ethylideneaminooxy!ethoxyl}benzyl)-thiazolidine-2,4-dione(hereinafter "Compound A"), 5-{4-(5-methoxy-3-methylimidazo5,4-b!pyridin-2-yl-methoxy)benzyl}thiazolidine-2,4-dione (preferably asits hydrochloride), 5-4-(6-methoxy-1-methylbenzimidazol-2-ylmethoxy)benzyl!thiazolidine-2,4-dione,5- 4-(1-methylbenzimidazol-2-ylmethoxy)benzyl!thiazolidine-2,4-dione and5-4-(5-hydroxy-1,4,6,7-tetramethyl-benzimidazol-2-ylmethoxy)benzyl!thiazolidine-2,4-dione.Troglitazone is disclosed in Japanese Patent Publication No. Hei 2-31079and in U.S. Pat. No. 4,572,912, and its formula is 5-{4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)methoxy!benzyl}-2,4-thiazolidinedione.Pioglitazone is disclosed in Japanese Patent Publication No. Sho62-42903 and No. Hei 5-66956 and in U.S. Pat. Nos. 4,287,200, 4,340,605,4,438,141, 4,444,779 and 4,725,610, and its formula is 5-{4-2-(5-ethylpyridin-2-yl)ethoxy!benzyl}-2,4-thiazolidinedione. Englitazoneis disclosed in Japanese Patent Publication No. Hei 5-86953 and in U.S.Pat. No. 4,703,052, and its formula is 5-3,4-dihydro-2-(phenylmethyl)-2H-benzo-pyran-6-ylmethyl!-2,4-thiazolidinedione.BRL-49653 is disclosed in Japanese Patent Kokai Application No. Hei1-131169 and in U.S. Pat. Nos. 5,002,953, 5,194,443, 5,232,925 and5,260,445, and its formula is 5-{4-2-methyl-2-(pyridin-2-ylamino)ethoxy!benzyl}-2,4-thiazolidinedione.Compound A is described in European Patent No. 708 098.5-{4-(5-Methoxy-3-methylimidazo5,4-b!pyridin-2-ylmethoxy)benzyl}-thiazolidine-2,4-dione (and itshydrochloride) are disclosed in Japanese Patent Kokai Application No.Hei 7-330728 and in European Patent No. 676 398. The above compounds maybe prepared as described in the prior art referred to above.5-(4-(6-Methoxy-1-methylbenzimidazol-2-ylmethoxy)benzyl!thiazolidine-2,4-dione,5- 4-(1-methylbenzimidazol-2-ylmethoxy)benzyl!thiazolidine-2,4-dione and5-4-(5-hydroxy-1,4,6,7-tetramethylbenz-imidazol-2-ylmethoxy)benzyl!thiazolidine-2,4-dioneare disclosed in European Patent Application No. 96303940.9, and may beprepared as described hereafter.

The active ingredients used in the present invention comprise, first,one or more HMG-CoA reductase inhibitors and, second, one or moreinsulin sensitizers or insulin resistance-improving agents. According tothe invention, a combination of the HMG-CoA reductase inhibitor and theinsulin sensitizer exhibits a synergistic effect in comparison with theapplication of the respective components alone, as shown below.Interestingly, such synergism appears to occur even if the compounds ofthe two classes do not always exist simultaneously in the body. That is,the synergistic effect may be observed even when the concentration ofone of the compounds of the two classes in the blood is less than thatrequired by itself to exhibit any appreciable effect. Although it is amere conjecture, it is thought that, when a compound of one of the twoclasses is received in the body and transported to a receptor, itactuates a "switch" in vivo. After some time, the level of the compoundin the blood may have decreased to a value at which it seems that nofurther effect should be observed, but the "switch" may still beactuated, thus maintaining the preventive and/or therapeutic effect forarteriosclerosis and/or xanthoma inherent in the compounds of thatclass. When a compound of the other class is administered to a patientin this state, the effect on the prevention and/or treatment ofarteriosclerosis and/or xanthoma may be combined with the effectresulting from the previous administration of the other compound, andthe effects of the two compounds operate together in a favourablesynergistic manner. It is, of course, obvious that it may well beconvenient to administer the two compounds simultaneously in clinicalpractice. Therefore, the HMG-CoA reductase inhibitor and the insulinsensitizer may be administered together in the form of a combinedpreparation. Alternatively, if it is difficult to mix the two agents,either because of some incompatibility between them or for some otherreason, for example problems in the mixing process, the two activeagents may be administered separately in the form of single doses. Asdescribed above, since the compounds of the two classes exhibit togethera synergistic effect, they may be administered almost simultaneously orat suitable intervals. The maximum interval acceptable for administeringthe compounds of the two classes in order to achieve the synergisticeffect of the present invention may be confirmed by clinical practice orby experiments using animals.

The HMG-CoA reductase inhibitors and insulin sensitizers of the presentinvention may generally be administered orally. Accordingly, thecompounds of the two classes may be separately prepared as two unitdosage forms or may be mixed physically to give a single unit dosageform. Examples of such formulations include, for example, powders,granules, tablets or capsules. These pharmaceutical formulations may beproduced by conventional means well known in the pharmaceutical field.

In the present invention, the individual doses of the HMG-CoA reductaseinhibitors and the insulin sensitizers and the ratio of between theamounts of the HMG-CoA reductase inhibitors and the insulin sensitizersmay vary widely, depending upon the activity of each compound and uponother factors, such as the condition, age and body weight of thepatient. For example, in the case of the insulin sensitizer, the potencyof BRL-49653 is about 100 times higher than that of troglitazone in vivoin a diabetic animal model, allowing the dose of these two compounds todiffer in theory by around two orders of magnitude, and, in practice, todiffer by around one order of magnitude. The dose of each of the HMG-CoAreductase inhibitors and the insulin sensitizers, where they are used inthe treatment of arteriosclerosis or xanthoma, would normally beexpected to be lower than that which is used when the two compounds areemployed separately for their original uses, that is asantihyperlipidemic and antidiabetic agents. Their doses are furtherlowered to some extent by the synergistic effect due to the combinationof the compounds of the two classes. For example, where pravastatin andtroglitazone are used in accordance with the invention, their dailydoses are preferably within the range of from 1 mg to 40 mg and from 1mg to 500 mg, respectively, as compared with doses of from 5 mg to 80 mgand from 10 mg to 1000 mg, respectively, where the compounds areemployed for their original uses as antihyperlipidemic and antidiabeticagents.

More generally, although, as remarked above, the dose of the HMG-CoAreductase inhibitors and the insulin sensitizers according to theinvention may widely vary, the daily dose is normally within the rangeof from 0.01 mg to 40 mg, preferably from 1 mg to 40 mg, and from 0.05mg to 500 mg, preferably from 1 mg to 500 mg, respectively.

The ratio between the compounds of these the two classes may also varywidely, however, we prefer that the ratio of the HMG-CoA reductaseinhibitor to the insulin sensitizer should be within the range of from1:200 to 200:1 by weight, preferably from 1:100 to 10:1 and morepreferably from 1:50 to 5:1 by weight.

The HMG-CoA reductase inhibitor and the insulin sensitizer in accordancewith the invention are preferably administered simultaneously or almostsimultaneously at a daily dose as described above, and may beadministered as a single dose or as divided doses.

The compounds and compositions of the present invention can beadministered in various forms, depending on the disease or disorder tobe treated and the age, condition and body weight of the patient, as iswell known in the art. For example, where the compounds or compositionsare to be administered orally, they may be formulated as tablets,capsules, granules, powders or syrups; or for parenteral administration,they may be formulated as injections (intravenous, intramuscular orsubcutaneous), drop infusion preparations or suppositories. Forapplication by the ophthalmic mucous membrane route, they may beformulated as eyedrops or eye ointments. These formulations can beprepared by conventional means, and, if desired, the active ingredientmay be mixed with any conventional additive, such as an excipient, abinder, a disintegrating agent, a lubricant, a corrigent, a solubilizingagent, a suspension aid, an emulsifying agent or a coating agent.

Examples of vehicles which may be employed include: organic vehiclesincluding; sugar derivatives, such as lactose, sucrose, glucose,mannitol and sorbitol; starch derivatives, such as corn starch, potatostarch, α-starch, dextrin and carboxymethylstarch; cellulosederivatives, such as crystalline cellulose, low-substitutedhydroxypropylcellulose, hydroxypropylmethylcellulose,carboxymethylcellulose, calcium carboxymethylcellulose and internallybridged sodium carboxymethylcellulose; gum arabic; dextran; Pullulane;and inorganic vehicles including silicate derivatives, such as lightsilicic anhydride, synthetic aluminum silicate and magnesium aluminatemetasilicate; phosphates, such as calcium phosphate; carbonates, such ascalcium carbonate; and sulfates, such as calcium sulfate.

Examples of lubricants which may be employed include: stearic acid;metal stearates, such as calcium stearate and magnesium stearate; talc;colloidal silica; waxes, such as beeswax and spermaceti wax; boric acid;adipic acid; sulfates, such as sodium sulfate; glycol; fumaric acid;sodium benzoate; DL-leucine; fatty acid sodium salts; lauryl sulfates,such as sodium lauryl sulfate and magnesium lauryl sulfate; silicates,such as silicic anhydride and silicic acid hydrate; and theaforementioned starch derivatives.

Examples of binders which may be employed include: polyvinylpyrrolidone;macrogol; and the same compounds as are mentioned above for thevehicles.

Examples of disintegrators which may be employed include: the samecompounds as are mentioned above for the vehicles; and chemicallymodified starches and celluloses, such as sodium crosscarmellose, sodiumcarboxymethylstarch and bridged polyvinylpyrrolidone.

Examples of stabilizers which may be employed include: paraoxybenzoates,such as methylparabene and propylparabene; alcohols, such aschlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkoniumchloride; phenols, such as phenol and cresol; thimerosal; dehydroaceticacid; and sorbic acid.

Examples of corrigents which may be employed include: sweetening agents,acidifiers and spices.

The present invention is further illustrated by the following Examples,which demonstrate the enhanced activity achieved by the synergisticcombination of the present invention. In addition, the subsequentFormulations illustrate the pharmaceutical formulations which may beprepared and the Preparations illustrate the preparation of certain ofthe insulin sensitizers used in the present invention.

EXAMPLE 1

WHHL rabbits 2-3 months of age, the Watanabe heritable hyperlipidemicrabbit described in Biochimica et Biophysica Acta, 960, 294-302 (1988)!were randomly assigned to a control group (7 animals, group A), a groupreceiving pravastatin alone (5 animals, Group B), a group receivingtroglitazone alone (7 animals, Group C), and a group receiving acombination of both active substances (6 animals, Group D). Pravastatinwas administered orally by gavage at a dose of 50 mg/kg/day once-dailyand troglitazone was given in the diet, containing 100 mg/kg of thesubstance for 32 weeks. The ingested amount was limited to a daily 120 gper rabbit. Blood was withdrawn from the animal immediately beforestarting the study and 4, 8, 12, 16, 20, 24, 28 and 32 weeks afterstarting the study and the total cholesterol levels (mg/dl) weredetermined for each blood sample. The levels are reported as apercentage (%) of the levels measured immediately before starting thestudy. The results are shown in Table 1. The animals were sacrificed andnecropsied at week 32 to examine (a) the percent lesion area (%) in thetotal, thoracic or abdominal portion of the aorta; (b) the stenosis (%)of the coronary arteries and (c) the incidence (%) of xanthoma in thedigital joints.

The results are shown in Table 2, Table 3 and Table 4. The valuesactually measured are represented as an average value±standard error inthose Tables.

                  TABLE 1                                                         ______________________________________                                        Week  Group A    Group B   Group C Group D                                    ______________________________________                                        0     (981 ± 25)*                                                                           (988 ± 19)*                                                                          (967 ± 54)*                                                                        (988 ± 47)*                                   (100)      (100)     (100)   (100)                                      4     103        87        88      70                                         8     102        87        89      69                                         12    98         81        78      66                                         16    98         81        83      65                                         20    90         75        72      57                                         24    83         68        73      59                                         28    79         68        77      61                                         32    76         60        76      61                                         ______________________________________                                         *actually measured volume (mg/dl)                                        

                  TABLE 2                                                         ______________________________________                                        Lesion area (%)                                                                      Total aorta                                                                            Thoracic aorta                                                                            Abdominal aorta                                   ______________________________________                                        Group A  65.7 ± 3.9                                                                            79.3 ± 5.4                                                                             29.9 ± 4.4                                 Group B  53.8 ± 8.2                                                                            64.6 ± 10.4                                                                            28.2 ± 8.1                                 Group C  51.7 ± 7.7                                                                            57.9 ± 9.9                                                                             27.6 ± 7.0                                 Group D  41.3 ± 7.7*                                                                           44.0 ± 9.5*                                                                            21.3 ± 7.3*                                ______________________________________                                         *p < 0.05. Significantly different against the control group under the        MannWhitney's UTest.                                                     

                  TABLE 3                                                         ______________________________________                                        Coronary stenosis (%)                                                               Number                                                                        of                                                                      Group animals  MLC     MRC   LAD  LCX   RCA  LSP                              ______________________________________                                        A     3        61      79    24   47    13   40                               B     2        71      81    16   34    18   9                                C     3        59      83    11   39    7    31                               D     3        39      81    3    23    1    27                               ______________________________________                                         MLC: main left coronary artery                                                MRC: main right coronary artery                                               LAD: left anterior descending artery                                          LCX: left circumflex artery                                                   RCA: right coronary artery                                                    LSP: left septal artery                                                  

                  TABLE 4                                                         ______________________________________                                        Incidence of xanthoma (%)                                                             Foreleg   Hind leg   Total                                            ______________________________________                                        Group A   100(14/14)  100(14/14) 100(28/28)                                   Group B   80(8/10)    80(8/10)   80(16/20)                                    Group C   86(12/14)   29(4/14)   57(16/28)                                    Group D   0(0/12)     0(0/12)    0(0/24)                                      ______________________________________                                    

As can be seen from the above example, no significant difference wasobserved in the change of plasma cholesterol levels at 32 weeks afteradministration between group D (which received a combination of bothagents) and group B (which received pravastatin alone). In contrast,there was observed clear synergism in the percent lesion area ratio(lesion area/total artery area in %) by comparing Group D (combinationtreatment) with Groups B and C (single agent treatment) as shown above.Synergism was observed in preventing coronary stenosis in respect of theleft anterior descending artery, the left circumflex artery and theright coronary artery. Development of xanthoma on the digital joints wasentirely prevented in Group D, thus demonstrating clear synergism.

Thus, although the levels of plasma cholesterol revealed no significantdifference when comparing the groups administered a combination of anHMG-CoA reductase inhibitor and an insulin sensitizer with the groupsadministered the active agent alone, the combination of both activeagents synergistically prevented progression of the arteriosclerosis,particularly of the thoracic aorta. These results could not be imaginedfrom the state of the prior art.

EXAMPLE 2

Male WHHL rabbits (2-3 months of age) which showed almost no arteriallesions were randomly assigned to a control group (7 animals, Group A),to a group subjected to oral administration of pravastatin alone (6animals, 50 mg/kg, group B), to a group subjected to oral administrationof pioglitazone alone (7 animals, 20 mg/kg, Group C), to a groupsubjected to oral administration of 5-(4-{2-1-(4-2'-pyridyl-phenyl)ethylideneaminooxy!ethoxy}benzyl)thiazolidine-2,4-dione(hereinafter Compound A, as described in EP 708 098, 7 animals, 10mg/kg, Group D), to a group subjected to oral administration of acombination of pravastatin and pioglitazone (6 animals, 50+20 mg/kg,Group E), and to a group subjected to oral administration of combinationof pravastatin and Compound A (7 animals, 50+10 mg/kg, Group F).

Each test compound was administered for eight months to the rabbits inthe form of an aqueous suspension (0.5% carboxymethylcellulose added).In the control group, a 0.5% carboxymethylcellulose solution only wasadministered. One month after the start of administration andthereafter, it was observed that the serum cholesterol in Groups B, Eand F was maintained at a lower level than that of the control group,and a 22 to 34% reduction of the serum cholesterol levels were observedin those groups. However, no reduction of the serum cholesterol levelswere observed in Groups C and D.

The percentage area covered by lesions at the aortic arch and over thewhole aorta are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Lesion Area (%)                                                               Group        Aortic Arch   Total Aorta                                        ______________________________________                                        A            82 ± 5(100)                                                                              59 ± 5(100)                                     B            59 ± 11(72)                                                                              *35 ± 6(70)                                     C            72 ± 10(88)                                                                              54 ± 8(108)                                     D            63 ± 9(77) 38 ± 8(76)                                      E            **43 ± 2(52)                                                                             *31 ± 4(62)                                     F            **33 ± 8(40)                                                                             **26 ± 5(52)                                    ______________________________________                                    

The values actually measured are represented by the mean value plus orminus standard error. The numbers in parentheses represent thepercentage lesion area against the control group.

*p<0.05, **p<0.01; significant difference against the control groupunder the Mann-Whitney's U-Test A significant difference (p<0.05) wasobserved between Groups B and F, between Groups C and E and betweenGroups D and F at the aortic arch, and between Groups C and E over thetotal aorta.

The average thickening of intima in aorta was measured and the resultsare shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Average thickening of intima (μm) in aorta                                 Group        Aortic Arch   Total Aorta                                        ______________________________________                                        A            237 ± 63(100)                                                                            154 ± 33(100)                                   B            194 ± 42(82)                                                                             126 ± 22(82)                                    C            245 ± 37(103)                                                                            177 ± 26(115)                                   D            291 ± 51(123)                                                                            162 ± 22(105)                                   E            189 ± 29(80)                                                                             118 ± 10(77)                                    F            146 ± 36(62)                                                                             94 ± 18(61)                                     ______________________________________                                    

The values actually measured are represented as the mean value plus orminus a standard error (μm). The numbers in parentheses represent thepercentage intimal thickening against the control group. A significantdifference was observed between Groups D and F at the aortic arch, andbetween Groups C and E and between Groups D and F over the total aorta(p<0.05) under the Mann-Whitney's U-Test.

The average thickening of the intima is calculated by thecross-sectional area of the aortic tunica intima one section from thearch and two sections from the thoracic and abdominal portions, dividedby the length of the tunica media.

A slight suppression of intimal thickening was observed in Group B,whilst no suppression of hypertrophy was observed in Groups C and D. Thesuppression of intimal thickening in Groups E and F was observed againstthat in Groups C and D.

The aortic cholesterol content was measured The tunica media and tunicaintima at the aortic arch and at the thoracic and abdominal aorta werepeeled away with tweezers and cut into pieces. The pieces were extractedwith a 2:1 by volume mixture of chloroform and methanol.

The chloroform phase was separated and evaporated to dryness and theresidue was dissolved in isopropanol. The total cholesterol and the freecholesterol were measured by a conventional enzymatic method.

The results are shown in Tables 7a and 7b.

                  TABLE 7a                                                        ______________________________________                                        Cholesterol Content of Aortic Arch                                            Group    Total       Free        Esterified                                   ______________________________________                                        A        27.1 ± 3.3(100)                                                                        20.6 ± 2.9(100)                                                                        6.5 ± 1.4(100)                            B        24.9 ± 5.2(92)                                                                         18.6 ± 4.4(90)                                                                         6.4 ± 1.1(98)                             C        33..5 ± 4.5(124)                                                                       26.9 ± 2.3(131)                                                                        6.6 ± 2.7(102)                            D        21.4 ± 0.7(79)                                                                         16.9 ± 1.8(82)                                                                         4.5 ± 1.3(69)                             E        24.3 ± 2.8(90)                                                                         18.0 ± 2.4(87)                                                                         6.3 ± 1.8(97)                             F        18.5 ± 2.6(68)                                                                         16.3 ± 2.5(79)                                                                         2.2 ± 0.8(34)*                            ______________________________________                                    

                  TABLE 7b                                                        ______________________________________                                        Cholesterol level of Thoracic and Abdominal Aorta                             Group    Total       Free        Esterified                                   ______________________________________                                        A        20.1 ± 2.3(100)                                                                        14.8 ± 2.3(100)                                                                        5.3 ± 1.7(100)                            B        17.2 ± 1.6(86)                                                                         12.5 ± 1.3(84)                                                                         4.7 ± 0.7(89)                             C        33.9 ± 7.3(169)                                                                        23.4 ± 3.8(158)                                                                        10.5 ± 3.8(198)                           D        14.0 ± 1.7(70)                                                                         9.2 ± 1.0(62)                                                                          4.9 ± 0.9(92)                             E        11.7 ± 2.2(58)*                                                                        8.5 ± 1.9(57)                                                                          3.2 ± 0.8(60)                             F        11.7 ± 1.7(58)**                                                                       7.8 ± 1.2(53)*                                                                         3.9 ± 0.7(74)                             ______________________________________                                         *p < 0.05;                                                                    **p < 0.02.                                                              

Data are expressed by the mean plus or minus standard error (mg/gtissue). The values in parentheses indicate the percentage against thecontrol. A significant difference against the control group was observedby the unpaired student t-test:

As is clear from Table 7, the total cholesterol levels of the thoracicand abdominal aorta are lower in Groups E and F than in Groups B, C andD. There was no clear cut trend between the free and esterifiedcholesterol levels. The results are similar to those of the rate oflesion area.

The incidence and the degree of xanthoma of the four legs were measured.The results are shown in Table 8.

                                      TABLE 8                                     __________________________________________________________________________                          Distribution of                                                                           Frequency of                                Incidence             severity of lesion                                                                        massive                                     Group                                                                              Forelegs                                                                           Hindlegs                                                                            Total -  +  ++ +++                                                                              xanthoma                                    __________________________________________________________________________    A    100  100   100(28/28)                                                                          0  12 10 6  57                                               (14/14)                                                                            (14/14)                                                             B    100.sup.*1)                                                                        75*.sup.*3)*4)                                                                      88.sup.*7)*8)                                                                       3  11 8  2  42.sup.*11)                                      (12/12)                                                                            (9/12)                                                                              (21/24)                                                       C    100  86.sup.*3)                                                                          93.sup.*9)                                                                          2  7  11 8  68.sup.*12)                                      (14/14)                                                                            (12/14)                                                                             (26/28)                                                       D    93.sup.*2)                                                                         86.sup.*6)                                                                          89.sup.*10)                                                                         3  19 6  0  21**.sup.*13)                                    (13/14)                                                                            (12/14)                                                                             (25/28)                                                       E    92   33**.sup.*3)*5)                                                                     63**.sup.*7)*9)                                                                     9  9  5  1  25*.sup.*12)                                     (11/12)                                                                            (4/12)                                                                              (15/24)                                                       F    50*.sup.*1)*2)                                                                     7**.sup.*4)*6)                                                                      29**.sup.*8)*10)                                                                    20 8  0  0  0**.sup.*11)*13)                                 (7/14)                                                                             (1/14)                                                                              (8/28)                                                        __________________________________________________________________________     Data in parentheses express the number of injured legs/number of examined     legs.                                                                         The severity of xanthoma was evaluated according to the following             criteria:                                                                     (-) no lesions                                                                (+) slight lesion                                                             (++) moderate lesion                                                          (+++) severe lesion                                                           *p < 0.05,                                                                    **p < 0.01. Significantly different from the control group.                   .sup.*1) p < 0.01. Significantly different between Groups B & F.              .sup.*2) p < 0.05. Significantly different between Groups D & F.              .sup.*3) p < 0.05. Significantly different between Groups B & E.              .sup.*4) p < 0.01. Significantly different between Groups B & F.              .sup.*5) p < 0.01. Significantly different between Groups C & E.              .sup.*6) p < 0.01. Significantly different between Groups D & F.              .sup.*7) p < 0.05. Significantly different between Groups B & E.              .sup.*8) p < 0.01. Significantly different between Groups B & F.              .sup.*9) p < 0.01. Significantly different between Groups D & F.              .sup.*10) p < 0.01. Significantly different between Groups D & F.             .sup.*11) p < 0.01. Significantly different between Groups B & F.             .sup.*12) p < 0.01. Significantly different between Groups C & E.             .sup.*13) p < 0.01. Significantly different between Groups D & F.        

As is clear from Table 8, the control group showed a 100% incidence ofxanthoma in all of the forelegs and hindlegs. Groups B, C and D showed aslightly less frequent incidence of xanthoma. Groups E and F, thecombination groups, showed a significantly lower frequency of incidenceof xanthoma. The trend is similar in the frequency of massive xanthoma,wherein the Groups E and F showed quite a low frequency of, or nooccurrence of, xanthoma, as opposed to Groups A to D.

The net results are that the two combinations of pravastatin, a HMG-CoAreductase inhibitor, and one of the thiazolidinedione insulinsensitizers exhibit synergistic effects on the treatment ofatherosclerosis and on the occurrence of xanthoma.

EXAMPLE 3

Synergism of HMG CoA reductase inhibitors and thiazolidinedione insulinsensitizers were examined on the regression of establishedatherosclerotic lesions in the cholesterol-fed rabbit model.

Male New Zealand white rabbits (5 months of age) were fed for two monthswith a 2% cholesterol diet, at the end of this time, the serumcholesterol of the rabbits increased to 1,100-4,100 mg/dl.

The rabbits were grouped randomly (3-9 animals per group) and the testsamples were administered orally for two months, while they were fedwith a normolipidaemic diet. The dosage of the test samples was: in thecase of pravastatin alone, 3 mg/kg or 5 mg/kg; in the case offluvastatin alone, 0.8 mg/kg or 1.5 mg/kg; in the case of troglitazonealone, 10 mg/kg; in the case of Compound A alone, 2.5 mg/kg. In the caseof combination groups, the dosage was: pravastatin 3 mg/kg+troglitazone10 mg/kg; pravastatin 5 mg/kg:+Compound A 2.5 mg/kg; fluvastatin 0.8mg/kg+troglitazone 10 mglkg; and fluvastatin 1.5 mg/kg+Compound A 2.5mg.

The results are shown in terms of the percentage of lesion area in thethoracic aorta.

                  TABLE 9                                                         ______________________________________                                                                          Rate of lesion                              Compound Dosage (mg/kg)                                                                            Number of animals                                                                          area (%)                                    ______________________________________                                        Control  (none)      3            29 ± 20 (100)                            Pravastatin                                                                            3           5            30 ± 12 (103)                                     5           5            23 ± 9 (79)                              Fluvastatin                                                                            0.08        5            27 ± 9 (93)                                       1.5         5            39 ± 16 (134)                            Troglitazone                                                                           10          6            23 ± 7 (79)                              Compound A                                                                             2.5         3            27 ± 7 (93)                              Pravastatin +                                                                           3 + 10     9            19 ± 5 (66)                              Troglitazone                                                                  Pravastatin +                                                                            5 + 2.5   5            9 ± 5 (31)                               Compound A                                                                    Fluvastatin +                                                                          0.8 + 10    7            18 ± 11 (62)                             Troglitazone                                                                  Fluvastatin +                                                                          1.5 + 2.5   5            18 ± 9 (62)                              Compound A                                                                    ______________________________________                                    

The values actually measured are expressed by the mean value±standarderror. The figures in parentheses represent percent reduction of lesionsagainst the control group.

As is clear from Table 9, each of the HMG CoA reductase inhibitors orthe thiazolidinedione insulin sensitizers alone showed no or littlereduction of the lesions, whilst all of the combination groups of thetwo components showed a synergistic reduction of the lesions.

EXAMPLE 4

Synergism of HMG CoA reductase inhibitors and thiazolidinedione insulinsensitizers was examined by another regression model, i.e. theregression of preformed atherosclerosis in hamsters. Male F_(l) bhamsters (weight about 130 g,) were given a diet containing 0.05%cholesterol for 13 weeks. They were grouped randomly (2-7 animals pergroup), and then the test samples were administered for 4 weeks whilethe hamsters were given a normolipidaemic diet. Pravastatin andfluvastatin were mixed with drinking water at the dose of 3 mg/kg and1.5 mg/kg, respectively, while troglitazone was mixed with the diet atthe dose of 30 mg/kg or 100 mg/kg.

In the case of the combination groups, the dosage was 3 mg/kg+30 mg/kgor 3 mg/kg+100 mg/kg for the pravastatin+troglitazone group, and 1.5mg/kg+30 mg/kg for the fluvastatin+troglitazone group.

The arterial lesions were evaluated by the extent of the area stainedwith Oil Red O (ORO), as described in Atherosclerosis, 114, 19-28(1995). Namely, the aortic arch was stained with ORO to prepare en facespecimens. The percentage area which was positive to the ORO stain overthe whole area was measured to represent the degree of aortic lesion.

After the treatment, the total serum cholesterol and triglyceride leveldid not significantly differ among the groups.

The results are shown in Table 10.

                  TABLE 10                                                        ______________________________________                                                Dosage              Stained Area                                      Compound                                                                              (mg/kg)  No of Animals                                                                            ORO (%)  % of Control                             ______________________________________                                        Control 0        5          1.82 ± 0.44                                                                         100                                      Pravastatin                                                                           3        5          1.93 ± 0.33                                                                         106                                      Fluvastatin                                                                           1.5      6          1.74 ± 0.49                                                                         96                                       Troglitazone                                                                          30       7          1.99 ± 0.40                                                                         109                                      Troglitazone                                                                          100      7          1.05 ± 0.64                                                                         58                                       Pravastatin +                                                                         3        5          1.28 ± 0.49                                                                         70                                       Troglitazone                                                                          30                                                                    Pravastatin +                                                                         3        4          0.63 ± 0.08                                                                         35                                       Troglitazone                                                                          100                                                                   Fluvastatin +                                                                         1.5      2          0.73     40                                       Troglitazone                                                                          30                                                                    ______________________________________                                    

There was a significant difference (p<0.05) between the control and thegroup receiving pravastatin+troglitazone 3 mg/kg+100 mg/kg and betweenthe group receiving pravastatin alone and the group receivingpravastatin+troglitazone 3 mg/kg+100 mg/kg.

As is clear from Table 10, no regression of aortic lesions was observedin the groups administered pravastatin, fluvastatin or troglitazone (30mg/kg) alone, although regression was observed with troglitazone aloneat the dosage of 100 mg/kg.

In the case of the combination of pravastatin and troglitazone,regression was observed, with a dose-dependent trend based ontroglitazone. In the case of the combination of fluvastatin andtroglitazone a similar synergistic regression of aortic lesions wasobserved.

In summary, it can be concluded that the combination of a HMG-CoAreductase inhibitor and a thiazolidinedione insulin sensitizer exhibit,as a class, both preventative and curative effects on atherosclerosisand on xanthoma.

PREPARATION 1 5-4-(1-Methylbenzimidazol-2-ylmethoxy)benzyl!-thiazolidine-2,4-dione

1(a) Methyl 4-nitrophenoxyacetate

A mixture of 56 g of 4-nitrophenol, 90 g of methyl bromoacetate, 100 gof potassium carbonate and 500 ml of dimethylformamide was stirred atroom temperature for 2 days. At the end of this time, the solvent wasremoved by distillation under reduced pressure. The resulting residuewas mixed with water and the aqueous mixture was extracted with ethylacetate. The extract was washed with water and dried over anhydroussodium sulfate, after which the solvent was removed by distillationunder reduced pressure. The resulting residue was triturated with hexaneto give 63.3 g of the title compound, melting at 98-99° C.

1(b) Methyl 4-aminophenoxyacetate

A solution of 30.8 g of methyl 4-nitrophenoxyacetate prepared asdescribed in step (a) above! in 500 ml of methanol was shaken in anatmosphere of hydrogen and in the presence of 5.0 g of 10 w/wpalladium-on-charcoal for 6 hours. At the end of this time, the reactionmixture was filtered and the filtrate was concentrated by evaporationunder reduced pressure, to give 25.8 g of the title compound having anRf value=0.79 (on thin layer chromatography on silica gel; developingsolvent: ethyl acetate).

1(c) Methyl 4-(2-bromo-2-butoxycarbonylethyl-1-yl)-phenoxyacetate

98 g of 47% w/W aqueous hydrobromic acid, followed by 33 ml of anaqueous solution containing 12.8 g of sodium nitrite, were added to asolution of 25.8 g of methyl 4-aminophenoxyacetate (prepared asdescribed in step (b) above! in 263 ml of a 2:5 by volume mixture ofmethanol and acetone, whilst ice-cooling, and the resulting mixture wasstirred, whilst ice-cooling, for 30 minutes. 18.2 g of butyl acrylatewere then added, and the reaction mixture was stirred for a further 30minutes, whilst ice-cooling. 3.2 g of copper(I) bromide were then addedto the mixture, and the mixture was stirred overnight at roomtemperature. At the end of this time, the reaction mixture was freedfrom the solvent by distillation under reduced pressure, and the residuewas mixed with an aqueous solution of sodium chloride. It was thenextracted with ethyl acetate. The extract was washed with an aqueoussolution of sodium chloride and dried over anhydrous sodium sulfate. Ondistilling off the solvent, there were obtained 51.7 g of the titlecompound having an Rf value=0.46 (on thin layer chromatography on silicagel; developing solvent: a 5:1 by volume mixture of hexane and ethylacetate) as a crude product.

1(d) 5- 4-(Ethoxycarbonylmethoxy)benzyl!thiazolidine-2,4-dione

A mixture of 100 g of methyl4-(2-bromo-2-butoxy-carbonylethyl-1-yl)phenoxyacetate prepared asdescribed in step (c) above!, 22 g of thiourea and 200 ml of ethanol washeated under reflux for 2.5 hours, after which 2N aqueous hydrochloricacid was added to the reaction mixture. The mixture was then heatedunder reflux for 5 hours. At the end of this time, the reaction mixturewas freed from the solvent by distillation under reduced pressure. Theresulting residue was diluted with water and the aqueous mixture wasextracted with ethyl acetate. The extract was dried over anhydrousmagnesium sulfate, after which the solvent was removed by distillationunder reduced pressure. The resulting residue was purified by columnchromatography through silica gel using a 2:5 by volume mixture of ethylacetate and hexane as the eluent, to give 19.4 g of the title compound,melting at 105°-106° C.

1(e) 5-4-(1-Methylbenzimidazol-2-ylmethoxy)benzyl!-thiazolidine-2,4-dione

A mixture of 1.0 g of N-methyl-1,2-phenylenediamine, 3.8 g of 5-4-(ethoxycarbonylmethoxy)benzyl!thiazolidine-2,4-dione prepared asdescribed in step (d) above!, 20 ml of concentrated aqueous hydrochloricacid, 10 ml of 1,4-dioxane and 10 ml of water was heated under refluxfor 5 hours. At the end of this time, the insoluble materials which hadprecipitated from the reaction mixture were collected by filtration andthe precipitate thus obtained was dissolved in tetrahydrofuran. Waterwas then added to the solution. The resulting aqueous mixture wasneutralized by adding sodium hydrogencarbonate and then extracted withethyl acetate. The extract was washed with an aqueous solution of sodiumchloride and dried over anhydrous sodium sulfate. The solvent was thenremoved by evaporation under reduced pressure, and the resulting residuewas purified by column chromatography through silica gel using ethylacetate and then ethanol as the eluent. The product was thenrecrystallized twice from a mixture of tetrahydrofuran and ethylacetate, to give 1.3 g of the title compound, melting at 230°-231° C.

PREPARATION 2 5-4-(6-Methoxy-1-methylbenzimidazol-2-ylmethoxy)-benzyl!thiazolidine-2,4-dione

2(a) 5-Methoxy-2-nitroaniline

70 ml of a 28% w/v methanolic solution of sodium methoxide were added atroom temperature to a solution of 25 g. of 5-chloro-2-nitroaniline in500 ml of 1,4-dioxane, and the resulting mixture was heated under refluxfor 4 hours, after which the solvent was removed by distillation underreduced pressure. The resulting residue was diluted with water, and theresulting aqueous mixture was extracted with ethyl acetate. The extractwas washed with a saturated aqueous solution of sodium chloride anddried over anhydrous sodium sulfate, after which the solvent was removedby distillation under reduced pressure. The resulting residue waspurified by column chromatography through silica gel using a gradientelution method, with mixtures of ethyl acetate and hexane in ratiosranging from 1:4 to 1:2 by volume as the eluent, to give 16.3 g of thetitle compound, melting at 124°-128° C.

2(b) N-t-Butoxycarbonyl-5-methoxy-2-nitroaniline

25 g of di-t-butyl dicarbonate, 15 ml of pyridine and 0.6 g of4-dimethylaminopyridine were added at room temperature to a solution of16 g of 5-methoxy-2-nitro-aniline prepared as described in step (a)above! in 500 ml of dehydrated tetrahydrofuran, and the resultingmixture was stirred for 2 hours. At the end of this time, the reactionmixture was freed from the solvent by distillation under reducedpressure, and the resulting residue was diluted with water. Theresulting aqueous mixture was extracted with ethyl acetate. The extractwas washed with a saturated aqueous solution of sodium chloride anddried over anhydrous sodium sulfate, after which the solvent was removedby distillation under reduced pressure. The resulting residue waspurified by column chromatography through silica gel using a 1:10 byvolume mixture of ethyl acetate and hexane as the eluent, to give 12.5 gof the title compound, melting at 112°-114° C.

2(c) N-t-Butoxycarbonyl-N-methyl-5-methoxy-2-nitroaniline

A solution of 49.6 g of N-t-butoxycarbonyl-5-methoxy-2-nitroanilineprepared as described in step (b) above! in 300 ml of dehydrateddimethylformamide was added, whilst ice-cooling, to a suspension of 12.0g of sodium hydride (as a 55% w/w dispersion in mineral oil) in 300 mlof dehydrated dimethylformamide, and the resulting mixture was stirredat room temperature for 30 minutes, after which 17.2 ml of methyl iodidewere added at room temperature. The reaction mixture was stirred for 1hour, after which it was allowed to stand overnight at room temperature.It was then concentrated to about one-fifth of its original volume byevaporation under reduced pressure. The concentrate was mixed withice-water and the resulting aqueous mixture was extracted with ethylacetate. The extract was washed with water and with a saturated aqueoussolution of sodium chloride, in that order, after which it was driedover anhydrous sodium sulfate. On distilling off the solvent, there wereobtained 52.1 g of the title compound, melting at 122°-124° C.

2(d) N-Methyl-5-methoxy-2-nitroaniline

750 ml of a 4N solution of hydrogen chloride in 1,4-dioxane were addedto 52 g of N-t-butoxycarbonyl-N-methyl-5-methoxy-2-nitroaniline preparedas described in step (c) above! at room temperature, and the resultingmixture was stirred for 2 hours. At the end of this time, the reactionmixture was freed from the solvent by distillation under reducedpressure, and the resulting residue was mixed with water and ethylacetate. The mixture was then neutralized by the addition of sodiumhydrogencarbonate, after which it was extracted with ethyl acetate. Theextract was washed with a saturated aqueous solution of sodium chlorideand dried over anhydrous sodium sulfate. On distilling off the solvent,there were obtained 35.3 g of the title compound, melting at 107°-110°C.

2(e) 5-Methoxy-N-methyl-1,2-phenylenediamine

346 g of stannous chloride were added to a mixture of 35 g ofN-methyl-5-methoxy-2-nitroaniline prepared as described in step (d)above!, 900 ml of t-butanol and 100 ml of ethyl acetate at roomtemperature, and the resulting mixture was stirred at 60° C. for 2hours, after which 11 g of sodium borohydride were added in portions at60° C. over a period of about 1 hour. The reaction mixture was thenstirred at 60° C. for 3 hours, after which it was allowed to stand atroom temperature for 2 days. It was then poured into ice-water and theaqueous mixture was neutralized by the addition of sodiumhydrogencarbonate. The mixture was extracted with ethyl acetate, and theextract was washed with a saturated aqueous solution of sodium chlorideand dried over anhydrous sodium sulfate. The solvent was removed fromthe mixture by distillation under reduced pressure, and the resultingresidue was purified by column chromatography through silica gel using a3:2 by volume mixture of ethyl acetate and hexane as the eluent, to give21.9 g of the title compound having an Rf value=0.18 (on thin layerchromatography on silica gel; developing solvent: a 1:1 by volumemixture of ethyl acetate and hexane).

2(f)5-(4-Methoxycarbonylmethoxybenzyl)-3-triphenyl-methylthiazolidine-2,4-dion

126 g of cesium carbonate were added at room temperature to a solutionof 120 g of 5-(4-hydroxy-benzyl)-3-triphenylmethylthiazolidine-2,4-dionein 2.5 litres of acetone, followed by 36 ml of methyl bromoacetate, andthe resulting mixture was stirred for 1 hour. At the end of this time,the reaction mixture was freed from the solvent by distillation underreduced pressure, and the resulting residue was mixed with water. Theaqueous mixture was then extracted with ethyl acetate. The extract waswashed with water and then with a saturated aqueous solution of sodiumchloride, after which it was dried over anhydrous magnesium sulfate. Thesolvent was removed by distillation under reduced pressure, after which1 litre of diethyl ether was added to the oily residue. The mixture wasthen agitated ultrasonically for 10 minutes. The solid substanceprecipitated was collected by filtration, to give 126.3 g of the titlecompound, melting at 158°-162° C.

2(g) 5-(4-Methoxycarbonylmethoxybenzyl)thiazolidine-2,4-dione

1700 ml of acetic acid and then 400 ml of water were added at roomtemperature to a suspension of 344 g of5-(4-methoxycarbonylmethoxybenzyl)-3-triphenylmethyl-thiazolidine-2,4-dioneprepared as described in step (f) above! in 400 ml of 1,4-dioxane andthe resulting mixture was stirred for 5 hours at 80° C. At the end ofthis time, the reaction mixture was freed from the solvent byevaporation under reduced pressure, and the resulting residue waspurified by column chromatography through silica gel using a 1:2 byvolume mixture of ethyl acetate and hexane, a 2 1 by volume mixture ofethyl acetate and hexane and then ethyl acetate as eluents, to give161.7 g of the title compound, melting at 100°-106° C.

2(h) 5-4-(6-Methoxy-1-Methylbenzimidazol-2-Ylmethoxy)-Benzyl!Thiazolidine-2,4-Dione

A mixture of 21.8 g of 5-methoxy-N-methyl-1,2-phenylenediamine preparedas described in step (e) above!, 63.4 g of5-(4-methoxycarbonylmethoxybenzyl)-thiazolidin-2,4-dione prepared asdescribed in step (g) above!, 250 ml of 1,4-dioxane and 750 ml ofconcentrated aqueous hydrochloric acid was heated under reflux for 60hours. At the end of this time, the reaction mixture was cooled withice, after which the solid matter was collected by filtration. 800 ml ofa 5% w/v aqueous solution-of sodum hydrogencarbonate was added to thismatter, and the resulting mixture was stirred at room temperature for 2hours. Insoluble materials were then collected by filtration anddissolved in a mixture of 1000 ml of dimethylformamide and 200 ml ofmethanol. The resulting solution was decolorized by treatment withactivated charcoal, which was then removed by filtration. The filtratewas then concentrated by evaporation under reduced pressure to a volumeof about 50 ml. The resulting concentrate was added to 750 ml of diethylether and the solution thus obtained was allowed to stand for 2 days. Atthe end of this time, the resulting precipitate was collected byfiltration, to give 20.1 g of the title compound, melting at 267°-271°C. and having an Rf value=0.68 (on thin layer chromatography on silicagel; using a developing solvent of methylene chloride containing 5% v/vethanol).

PREPARATION 3 5-4-(5-Hydroxy-1,4,6,7-tetramethylbenzimidazol-2-yl-methoxy)Benzyl!thiazolidine-2,4-dione

3(a) Trimethylbenzoquinone

A suspension of 25.6 g of ferric chloride in 50 ml of water was added atroom temperature to a solution of 20 g of trimethylhydroquinone in 150ml of acetone, and the resulting mixture was stirred for 1 hour, afterwhich it was allowed to stand for 2 days. At the end of this time, itwas concentrated to about one half of its original volume, and theconcentrate was mixed with water. The resulting aqueous mixture wasextracted with ethyl acetate, and the extract was washed with water andwith a saturated aqueous solution of sodium chloride, in that order,after which it was dried over anhydrous sodium sulfate. The solvent wasremoved by distillation under reduced pressure, and the resultingresidue was purified by column chromatography through silica gel, usinga 1:6 by volume mixture of ethyl acetate and hexane as the eluent, togive 16.9 g of the title compound having an Rf value=0.48 (on thin layerchromatography on silica gel; developing solvent: a 1:6 by volumemixture of ethyl acetate and hexane).

3(b) 2,3,6-Trimethylbenzoauinone-4-oxime

A solution of 7.04 g of hydroxylamine hydrochloride in 30 ml of waterwas added at room temperature to a solution of 16.9 g oftrimethylbenzoquinone prepared as described in step (a) above! in 150 mlof methanol, and the resulting mixture was stirred for 2 hours, afterwhich it was allowed to stand for 2 days. At the end of this time, thereaction mixture was diluted with 1000 ml of water. The precipitatewhich separated out was collected by filtration and recrystallized froma mixture of ethyl acetate and hexane, to give 11.2 g of the titlecompound, melting at 188°-190° C.

3(c) 4-Hydroxy-2,3,5-trimethylaniline

152 g of sodium hydrosulfite were added, whilst ice-cooling, to amixture of 36.15 g of 2,3,6-trimethyl-benzoquinone-4-oxime prepared asdescribed in step (b) above! and 880 ml of a 1N aqueous solution ofsodium hydroxide, and the resulting mixture was stirred at roomtemperature for 1 hour, after which it was allowed to stand overnight.The reaction mixture was then poured into ice-water and the pH of theaqueous mixture was adjusted to a value of 4 to 5 by the addition of 5Naqueous hydrochloric acid, after which it was neutralized with sodiumhydrogencarbonate. The mixture thus obtained was extracted with ethylacetate. The extract was washed with a saturated aqueous solution ofsodium chloride and dried over anhydrous sodium sulfate. The solvent wasthen removed by distillation under reduced pressure, after which thecrystalline residue was triturated with diisopropyl ether and collectedby filtration. On washing with diisopropyl ether, there were obtained30.1 g of the title compound, melting at 131°-134° C.

3(d) N-t-Butoxycarbonyl-4-hydroxy-2,3,5-trimethylaniline

22.0 ml of triethylamine were added at room temperature to a solution of20 g of 4-hydroxy-2,3,5-trimethylaniline prepared as described in step(c) above! in 500 ml of tetrahydrofuran, followed by 34.6 g ofdi-t-butyl dicarbonate, and the resulting mixture was stirred for 6hours, after which it was allowed to stand overnight. At the end of thistime, the reaction mixture was freed from the solvent by distillationunder reduced pressure, and the resulting residue was mixed with water.The resulting aqueous mixture was extracted with ethyl acetate. Theextract was washed with a saturated aqueous solution of sodium chlorideand dried over anhydrous sodium sulfate. The solvent was removed bydistillation under reduced pressure, after which the crystalline residuewas triturated with hexane, to give 31.9 g of the title compound,melting at 158°-161° C.

3(e) N-Methyl-4-hydroxy-2,3,5-trimethylaniline

A solution of 15 g ofN-t-butoxycarbonyl-4-hydroxy-2,3,5-trimethylaniline prepared asdescribed in step (d) above! in 200 ml of dehydrated tetrahydrofuran wasadded to a suspension of 6.8 g of lithium aluminum hydride in 300 ml ofdehydrated tetrahydrofuran, whilst ice-cooling, and the resultingmixture was stirred at room temperature for 3 hours, after which it washeated under reflux for 2 hours. At the end of this time, a mixture of10 ml of water and 30 ml of tetrahydrofuran was added to the reactionmixture in order to destroy any excess of lithium aluminum hydride. Thereaction mixture was then stirred at room temperature for 1.5 hours,after which insoluble materials were filtered off with the aid of aCelite (trade mark) filter aid. These materials were washed with ethylacetate, and the ethyl acetate washings were combined and dried overanhydrous sodium sulfate. The solvent was removed by distillation underreduced pressure, and the resulting residue was purified by columnchromatography through silica gel using a 1:3 by volume mixture of ethylacetate and hexane as the eluent, to give 5.1 g of the title compound,melting at 120°-122° C.

3(f) N-t-Butoxycarbonyl-N-methyl-4-hydroxy-2,3,5-trimethylaniline

5.0 ml of triethylamine and a solution of 7.92 g of di-t-butyldicarbonate in 30 ml of tetrahydrofuran were added at room temperatureto a solution of 5.0 g of N-methyl-4-hydroxy-2,3,5-trimethylanilineprepared as described in step (e) above! in 70 ml of tetrahydrofuran,and the resulting mixture was stirred for 1 hour, after which it wasallowed to stand overnight. At the end of this time, the reactionmixture was freed from the solvent by distillation under reducedpressure, and the resulting residue was mixed with water. The aqueousmixture was extracted with ethyl acetate. The extract was washed withwater and with a saturated aqueous solution of sodium chloride, in thatorder, after which it was dried over anhydrous magnesium sulfate. Afterdistilling off the solvent, the residual crystals were triturated withhexane and collected by filtration. There were obtained 7.35 g of thetitle compound, melting at 163°-166° C.

3(g) N-t-Butoxycarbonyl-N-methyl-4-acetoxy-2,3,5-trimethylaniline

5.64 ml of dehydrated triethylamine and 2.9 ml of acetyl chloride wereadded at room temperature to a solution of 7.2 g ofN-t-butoxycarbonyl-N-methyl-4-hydroxy-2,3,5-trimethylaniline prepared asdescribed in step (f) above! in 100 ml of dehydrated tetrahydrofuran,and the resulting mixture was stirred for 1 hour, after which it wasallowed to stand overnight. The reaction mixture was then diluted withwater and the aqueous mixture was extracted with ethyl acetate. Theextract was washed with water and with a saturated aqueous solution ofsodium chloride, in that order, after which it was dried over anhydrousmagnesium sulfate. The solvent was removed by distillation under reducedpressure, after which the residue was triturated with ice-cooled hexaneto cause crystallization. The crystals were collected by filtration andwashed with ice-cooled hexane to give 6.25 g of the title compound,melting at 103°-104° C.

3(h) N-Methyl-4-acetoxy-2,3,5-trimethylaniline Hydrochloride

A mixture prepared by adding 100 ml of a 4N solution of hydrogenchloride in 1,4-dioxane to 5.45 g ofN-t-butoxycarbonyl-N-methyl-4-acetoxy-2,3,5-trimethyl-aniline preparedas described in step (g) above! at room temperature was stirred for 3hours. At the end of this time, the reaction mixture was freed from thesolvent by distillation under reduced pressure, and the resultingresidue was triturated with diisopropyl ether. The crystals thusobtained were collected by filtration, after which they were washed withdiisopropyl ether to give 4.36 g of the title compound, melting at172°-176° C.

3(i) N-Methyl-4-acetoxy-2,3,5-trimethyl-6-nitroaniline

4.3 g of N-methyl-4-acetoxy-2,3,5-trimethylaniline hydrochlorideprepared as described in step (h) above! were added to ice-cooledconcentrated aqueous nitric acid, and the resulting mixture was stirred,whilst ice-cooling, for 10 minutes and then at room temperature for 10minutes. At the end of this time, the reaction mixture was poured intoice-water and the aqueous mixture was neutralized by the addition ofsodium hydrogencarbonate, after which it was extracted with ethylacetate. The extract was washed with a saturated aqueous solution ofsodium chloride and dried over anhydrous sodium sulfate. The solvent wasthen removed by distillation under reduced pressure, after which 50 mlof diisopropyl ether and 50 ml of hexane were added to the residue. Themixture was then agitated ultrasonically for 5 minutes. Insolubleprecipitates were triturated with a 1:1 by volume mixture of diisopropylether and hexane. The resulting crystals were collected by filtration,after which they were washed with a 1:1 by volume mixture of diisopropylether and hexane to give 2.76 g of the title compound, melting at143°-146° C.

3(j) 4-Acetoxy-N-Methyl-3,5,6-trimethyl-1,2-phenylene-diamine

A solution of 2.65 g ofN-methyl-4-acetoxy-2,3,5-trimethyl-6-nitroaniline prepared as describedin step (i) above! in a mixture of 20 ml ethanol and 20 ml of ethylacetate was shaken at room temperature for 3.5 hours and then at 40° C.for 3 hours in an atmosphere of hydrogen and in the presence of 0.2 g ofplatinum oxide. At the end of this time, the reaction mixture wasfiltered to remove the platinum oxide and the filtrate was freed fromthe solvent by distillation under reduced pressure. The resultingresidue was purified by column chromatography through silica gel, usinga 1:1 by volume mixture of ethyl acetate and hexane as the eluent, togive 1.3 g of title compound, melting at 113°-116° C.

3(k) 5- 4-(5-Hydroxy-1,4,6,7-tetramethylbenzimidazol-2-ylmethoxy)benzyl!Thiazolidine-2,4-Dione

A mixture of 1.0 g of4-acetoxy-N-methyl-3,5,6-trimethyl-1,2-phenylenediamine prepared asdescribed in step (j) above!, 2.7 g of5-(4-methoxycarbonylmethoxy-benzyl)thiazolidine-2,4-dione prepared asdescribed in step 2(g) of Preparation 2!, 5 ml of 1,4-dioxane and 25 mlof concentrated aqueous hydrochloric acid was heated under reflux for 2days. At the end of this time, the reaction mixture was added toice-water and the resulting mixture was neutralized by the addition ofsodium hydrogencarbonate. It was then extracted with ethyl acetate. Theextract was washed with a saturated aqueous solution of sodium chlorideand dried over anhydrous magnesium sulfate. The solvent was then removedby distillation under reduced pressure, after which the residue waspurified by column chromatography through silica gel, using ethylacetate as the eluent.

Fractions containing the title compound were collected and the solventwas removed by distillation under reduced pressure, to give a redresidual oil. 150 ml of diethyl ether were added to the oil, and themixture was agitated ultrasonically for 5 minutes. The precipitate whichseparated out was collected by filtration and dissolved in 300 ml oftetrahydrofuran. The resulting solution was concentrated to a volume ofbetween about 10 ml and 20 ml by evaporation under reduced pressure. 200ml of ethyl acetate were added to the concentrate, and the mixture wasagitated ultrasonically for 20 minutes. The precipitate which separatedout was collected by filtration, to give 0.52 g of the title compound,melting at 240°-244° C. and having an Rf value=0.44 (on thin layerchromatography on silica gel; developing solvent: ethyl acetate).

FORMULATION 1

Capsules

0.5 g of pravastatin sodium, 20 g of troglitazone, 1.5 g of crospovidone(polyvinylpyrrolidone disintegrator) and 0.2 g of sodium lauryl sulfatewere blended. The mixture was divided between 100 empty capsules(number 1) to give 100 capsules, each containing 5 mg of pravastatinsodium and 200 mg of troglitazone.

FORMULATION 2

Tablets

40 g of a 5% w/v aqueous solution of hydroxypropyl-cellulose were addedto a mixture of 5 g of pravastatin sodium, 2 g of Compound A, 24 g ofhydroxypropyl-cellulose (low degree of substitution) and 86.9 g oflactose, and the resulting mixture was kneaded to give a composition.This composition was passed through a 10 mesh (Tyler standard mesh)screen and dried, after which it was passed through a 15 mesh (Tylerstandard mesh) screen to give even sized grains. 11.9 g of the grainsand 0.1 g of magnesium stearate were mixed and the mixture was made intotablets with a tableting machine, giving tablets of 6.5 mm diameter and120 mg weight, each containing 5 mg of pravastatin sodium and 2 mg ofCompound A.

We claim:
 1. A method for the prevention or treatment ofarteriosclerosis or xanthoma, which method comprises administering to apatient suffering from or susceptible to arteriosclerosis or xanthoma afirst agent selected from the group consisting of HMG-CoA reductaseinhibitors and a second agent selected from the group consisting ofinsulin sensitizers, said first and second agents being administeredtogether in a synergistic mixture or individually in amounts and withinsuch a period as to act synergistically together;wherein said HMG-CoAreductase inhibitor is pravastatin and said insulin sensitizer istroglitazone.
 2. The method of claim 1 wherein 0.01 mg to 40 mg ofpravastatin and 0.05 mg to 500 mg of troglitazone are administered. 3.The method of claim 1 wherein 1 mg to 40 mg of pravastatin and 1 mg to500 mg of troglitazone are administered.
 4. The method of claim 2wherein the ratio of amounts of pravastatin to troglitazone administeredis 1:200 to 200:1.
 5. The method of claim 4 wherein the ratio of amountsof pravastatin to troglitazone administered is 1:100 to 10:1.
 6. Themethod of claim 5 wherein the ratio of amounts of pravastatin totroglitazone administered is 1:50 to 5:1.